BET (bromo-domain and extra-terminal domain) family proteins have garnered tremendous interest as pharmacologic targets for the treatment of benign and malignant diseases, however their mechanisms of actions are not well understood. In previous studies, we found that the hematopoietic transcription factor GATA1 is acetylated leading to recruitment of BETs. BET proteins are highly co-localized with GATA1 genome-wide and function at multiple steps of the transcription cycle. In the following specific aims, we will study the major expressed BETs, Brd2, Brd3, and Brd4 with regards to their individual and overlapping functions, molecular mechanisms of action, their roles as epigenetic conveyors of cellular mitotic memory, and their role in higher order chromatin organization. Aim 1: Shared and specific functions of BETs. Using genomic surveys, phenotypic knockout-rescue assays, and a new gene-specific tethering approach, we will functionally and mechanistically dissect BET protein function. Aim 2: Mitotic bookmarking by BETs. During mitosis transcription is globally disrupted due to eviction of the majority of nuclear factors from chromatin. This originated the idea that proteins that are retained on mitotic chromosomes, such as BETs, might serve as mitotic bookmarks to ensure faithful transcription reactivation. We will test and functionally dissect potential mitotic bookmarking function of BETs. Aim 3: linking Brd2 and CTCF in chromatin organization. Our preliminary data show that Brd2 co-localizes to a high degree with the architectural nuclear factor CTCF genome-wide, suggesting a role of BET proteins in the regulation of chromosome structure. We will examine the functional interplay between Brd2 and CTCF with regards to gene expression and higher order chromatin architecture. The work proposed here is aimed at obtaining deep insights into the regulation of hematopoietic gene expression with focus on BET proteins. Our studies benefit from a host of data sets, tools and reagents that we generated that uniquely enable us to examine mechanisms of BET action and better understand pharmacologic BET inhibitors.